Lipids containing omega-3 and omega-6 fatty acids

ABSTRACT

A lipid preparation including a glycerophospholipid or salt, conjugate and derivatives thereof, particularly phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl-inositol (PI), phosphatidylglycerol (PG) and phosphatidic acid (PA), and poly-unsaturated fatty acid (PUFA) acyl groups, particularly long-chain poly-unsaturated fatty acid (LC-PUFA) acyl groups such as omega-3 and/or omega-6 acyl groups, wherein said PUFA is covalently bound to said glycerophospholipid. The preparation possesses an improved bioactivity, and is useful in the treatment of various cognitive and mental conditions and disorders and for maintenance of normal functions of brain-related systems and processes.

RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 10/994,175, filed Nov. 19, 2004, which in turn is acontinuation of International Patent Application No. PCT/IL2004/000957,filed Oct. 21, 2004, the contents of which are here incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to phospholipids and polar lipidspreparations which are enriched with omega-3 and/or omega-6 fatty acidscovalently attached to the lipid backbone. The phospholipid preparationsof the invention are particularly useful as nutraceuticals, foodadditives and/or pharmaceutical agents for the treatment of variousconditions, in particular related to cognitive functions.

2. Prior Art

Lipids, and especially polar lipids, nitrogen containing lipids, andcarbohydrate containing lipids (phospholipids, sphingosines,glycolipids, ceramides, sphingomyelins) are the major building blocks ofcell membranes, tissues, etc. Additionally they play important roles insignal transduction processes and in a variety of biochemical andbiosynthetic pathways.

Glycerophospholipids, lipids based on a glycerol backbone and containinga phosphate head group, are the main building blocks of cell membranes.Since most, if not all, biochemical processes involve cell membranes,the structural and physical properties of membranes in different tissuesis crucial to the normal and efficient functioning of membranes in allbiochemical processes.

In light of the emerging functional foods category in the area ofdietary lipids many health benefits have been attributed to theconsumption of certain fatty acids. For example, it has been reported inmany research studies that polyunsaturated fatty acids (PUFA) of thetype omega-3 and omega-6, have several health benefits on cardiovasculardisease, immune disorders and inflammation, renal disorders, allergies,diabetes, and cancer. These types of fatty acids are naturally occurringmainly in fish and algae, where they are randomly distributed on thesn-1, sn-2, and sn-3 positions of the glycerol backbone oftriglycerides.

The professional literature emphasizes the importance of an adequatediet containing omega-3 fatty acids. Extensive clinical studiesinvestigating the importance of Docosahexaenoic acid (DHA), one of themost important omega-3 fatty acids, in the brain, found that low levelsof DHA are associated with depression, memory loss, dementia, and visualproblems. All studies showed a dramatic improvement in the elderly brainfunction as blood levels of DHA increased.

Other known benefits of DHA include: lower risk of arrhythmias,reduction in the risk of sudden cardiac death, lower plasma triglyceridelevels and reduced blood clotting tendency. Furthermore, DHA may haveimportance in the field of brain functioning enhancement, baby formulafortification, diabetics and cancer. Nutritional studies, investigatingthe importance of DHA in the brain, found that low levels of DHA areassociated with depression, memory loss, cognitive impairment, dementiaand visual problems.

The human body does not adequately synthesize DHA. Therefore it isnecessary to obtain it from the diet. Humans obtain DHA from theirdiets, initially through the placenta, then from breast milk, and laterthrough dietary sources, such as fish, red meats, animal organ meats andeggs. Popular fish like tuna, salmon and sardines are rich sources.Until recently, the primary source of DHA dietary supplements has beenfish oils. The ability of enzymes to produce the omega-6 and omega-3family of products of linoleic and alpha-linolenic acid declines withage. Because DHA synthesis declines with age, as we get older our needto acquire DHA directly from diet or supplements increases. In fact,several recent publications suggested DHA to be considered as essentialfatty acid [for example, Muskiet, F. et al. (2004) J Nutr.134(1):183-6].

Because DHA is important for signal transmission in the brain, eye andnervous system, many consumers concerned with maintaining mental acuityare searching for a pure, safe way to supplement their DHA levels.

Polyunsaturated acids, in particular long chain, such as omega-3 and 6,have been shown to confer many valuable health benefits on thepopulation. The global market for long-chain PUFAs, including the foodsegment, is rapidly growing.

The majority of efforts in the industry are however invested in theimprovement of PUPA processing techniques and in the creation or higherconcentrated grades of PUFA derivatives to accommodate dietarysupplements and functional foods needs.

The academic and industrial communities are less concerned regarding theevaluation of different delivery approaches of PUFA in order to enhancetheir bio-availability and their efficacy in term of their known varietyof health benefits. These benefits range from prevention and treatmentof CVD, diabetes, cognitive disorders and/or decline, visual disorders,skin conditions, learning disorders, etc. Additionally, PUFAs have beenshown to assist in the cognitive and visual development of infants.

SUMMARY OF THE INVENTION

PUFA-Lipids

PS-PUFA

Phosphatidylserine, also known as PS, is a natural phospholipid withbio-functionality that has made it one of the most promising dietarysupplements in the field of brain nutrition. PS and its health benefitshave been known to the scientific and nutrition communities since the1970's. Numerous studies have been conducted in order to establish thisefficacy in a variety of cognitive and mental functions. Those studieshave shown that PS can improve memory, fight dementia, fight earlystages of Alzheimer's disease, reduce stress and tension, improveattention span, enhance mood and fight depression, to name but few.

PS is one of the most important building blocks of cell membranes in thebrain. Hence, the level of PS in brain cell membranes ensures thefluidity and structure of these membranes. The normal level ensuresnormal and efficient signal transduction processes, efficient glucoseconsumption, and other biological pathways that result in normalcognitive and mental functions.

Since PS is not abundant in human nutrition and since in many people,especially the elderly, the biosynthetic pathways responsible for theproduction of PS are malfunctioning, the levels of PS in the body andbrain are low. This results in a variety of cognitive and mentaldisorders, such as depression, memory loss, short attention span,learning difficulties, etc.

The supplementation of PS in the diets of elderly people with suchdisorders has resulted, in many cases, in dramatic improvements of thesedisorders. Over the recent years, studies have shown that even youngerpeople can benefit from dietary supplementation of PS. PS has been shownto improve the learning capabilities of students, improve memory andattention span, etc.

It is therefore an object of the present invention to provide specialpreparations of PS, for use mainly as nutraceuticals and as functionalfood additives.

PC-PUFA

As mentioned before, phospholipids are essential components of allcellular and sub-cellular membranes. Phosphatidylcholine andphosphatidylethanolamine predominate quantitatively, substantiallyconstituting the typical bilayer configuration. Phospholipids belong tothe amphipathic molecules with a water-soluble and a fat-solublecomponent. In the bilayer configuration the hydrophilic groups arearranged at the outer and inner side of the membrane toward thesurrounding medium; the lipophilic groups, in contrast, face each otherat the inner side of the bilayer configuration.

Other important constituents of biological membranes are cholesterol,glycolipids, and peripheral and integral proteins. The basic structureof biological membranes is thus a series of recurrent unities oflipid-protein complexes. The membrane is asymmetric. The function of theexternal (cellular) and internal (sub cellular) membrane systems dependson their composition and on the integrity of their phospholipidstructure. In addition to their presence in cell membranes,phospholipids constitute structural and functional elements of thesurface mono-layers of lipoproteins and of surfactants.

Of utmost importance for the function of biological membranes is theirfluidity, which is decisively influenced by phospholipids. Besides thecontent in cholesterol and proteins and the nature and charge of thepolar head groups of phospholipids in the system, membrane fluiditydepends on the length of the chains of fatty acid residues in thephospholipid molecule, as well as on the number and type of pairing oftheir double bonds.

Phospholipids containing poly-unsaturated fatty acids supply theorganism with important building blocks which improves membranefluidity.

Studies conducted with PUFA-containing phospholipids have shown thefollowing:

1. They are high-energy, basic, structural, and functional elements ofall biological membranes, such as cells, blood corpuscles, lipoproteins,and the surfactant.

2. They are indispensable for cellular differentiation, proliferation,and regeneration.

3. They maintain and promote the biological activity of manymembrane-bound proteins and receptors.

4. They play a decisive role for the activity and activation of numerousmembrane-located enzymes, such as sodium-potassium-ATPase, adenylatecyclase and lipoprotein lipase.

5. They are important for the transport of molecules through membranes.

6. They control membrane-dependent metabolic processes between theintracellular and intercellular space.

7. The polyunsaturated fatty acids contained in them, such as linoleicacid, are precursors of the cytoprotective prostaglandins and othereicosanoids.

8. As choline and fatty acid donors they have an influence in certainneurological processes.

9. They emulsify fat in the gastrointestinal tract.

10. They are important emulsifiers in the bile.

11. They codetermine erythrocyte and platelet aggregation.

12. They influence immunological reactions on the cellular level.

Phospholipids containing PUFA are theoretically of importance in allthose diseases in which damaged membrane structures, reducedphospholipid levels, and/or decreased membrane fluidity are present.This hypothesis is supported by experimental and clinical investigationsof various membrane-associated disorders and illnesses.

Studies on the active principle as well as pharmacological and clinicaltrials are available on a variety of disturbances and diseases relatedto membrane damages. For example, in liver diseases the hepatocytestructures are damaged by, for example, viruses, organic solvents,alcohol, medicaments, drugs, or fatty food. As a consequence, membranefluidity and permeability may be disturbed, and membrane-dependentmetabolic processes as well as membrane-associated enzyme activities maybe impaired. This considerably inhibits the metabolism of the liver.

Other examples include hyperlipoproteinemia with or withoutatherosclerosis, hemorrheological disturbances with an elevatedcholesterol/phospholipid ratio in the membranes of platelets and redblood cells, neurological diseases, gastro intestinal inflammations,kidney diseases, and in a variety of aging symptoms.

All these very different diseases have in common comparable membranedisorders. With polyunsaturated phosphatidylcholine molecules suchdisorders may be positively influenced, eliminated, or even improvedbeyond normal due to the high content in polyunsaturated fatty acids.Following are some examples of the mechanisms that mediate thisphenomenon:

1. HDL particles enriched with PUFA-containing-phosphatidylcholine areable to take up more cholesterol from low-density lipoprotein (LDL) andtissues. More cholesterol can be transported back to the liver. Thisaction on the cholesterol reverse transport is unique. All otherlipid-lowering agents reduce either the cholesterol absorption in thebody or the cholesterol synthesis in the liver and its distribution tothe periphery. These substances, however, do not physiologicallymobilize the cholesterol already present in the periphery.

2. The cholesterol/phospholipid ratio in membranes, platelets, and redblood cells decreases and membrane function is improved up tonormalization.

3. Peroxidative reactions are reduced, damaged hepatocyte membranestructures restored, membrane fluidity and function stabilized,immuno-modulation and cell protection improved, and membrane-associatedliver functions enhanced.

4. With the normalization of the cholesterol/phospholipid ratio, thebile is also stabilized.

5. Due to its specific property as a surface-active emulsifier,PUFA-containing-phosphatidylcholine solubilize fat and is used inreducing the risk and treatment of fat embolism.

6. The substitution with poly-unsaturated-fatty-acids and choline mayhave a cytoprotective effect in the brain and activate neuronalprocesses.

7. Liposomes with polyunsaturated phosphatidylcholine molecules may actas drug carriers, such as of vitamin E.

Liver Disease

Experimental and clinical results support the assumption that thetherapeutic application of PUFA-containing-phosphatidylcholine hasprotective and even curative and regenerative effects on biologicalmembranes of sinus endothelial cells and hepatocytes. The cytoprotectiveeffect of PUFA-containing-phosphatidylcholine has been corroborated in 7in vitro and in 55 in vivo experiments, in which 20 different modelswith five different animal species were used. Types of intoxication thatare known to play a role in the etiology of liver disease have mostlybeen applied: chemical substances, medicaments, alcohol, cholestasis,immunological phenomena, exposure to radiation, and so on.

The hepato-protective effects of PUFA-containing-phosphatidylcholinehave been confirmed and were the more pronounced the earlierPUFA-containing-phosphatidylcholine was administered:

1. Structures of membranes were normal or largely normalized.

2. Fatty infiltrations and hepatocyte necrosis could be diminished oreven eliminated.

3. Corresponding data were found for lipid peroxidation, transaminaseand cholinesterase activity, and for serum lipids; liver cell metabolismincreased.

4. The increase of RNA and protein synthesis and of the liver cellglycogen content indicated a stimulation of the liver cells.

5. Reduced collagen production, collagen/DNA ratio, and liverhydroxyproline content indicated a reduced formation of connectivetissue.

The dosage of PUFA-containing-phosphatidylcholine ranged from 525 to2,700 mg/day when administered orally, and from 500 to 3,000 mg/day inintravenous application. The duration of treatment lasted from a fewweeks to up to 30 months. The main liver indications were acutehepatitis, chronic hepatitis, fatty liver, toxic liver damage, cirrhosisof the liver, and hepatic coma.

The clinical findings, showing the effectiveness ofPUFA-containing-phosphatidylcholine, can be summarized generally asfollows:

1. Accelerated improvement or normalization of subjective complaints, ofclinical findings, and of several biochemical values

2. Better histological results as compared with the control groups

3. A shortened duration of hospitalization

Promising results were obtained also in renal disorders, chronicambulatory peritoneal dialysis, hyperlipoproteinemia/atherosclerosis,gastrointestinal inflammation, psoriasis, and more.

Recent research studies have shown that PUFA-enriched phospholipids,isolated from rainbow trout embryos, have novel health benefits. Some ofthese benefits include the treatment of tumor cells, inhibition of5-lipoxygenase activity, reduction of neutral fat levels (such ascholesterol).

There is proof that a person who receives enriched phospholipidsnutritionally, these phospholipids cross the intestinal barrier and theblood-brain barrier, thus reaching the brain. Recently, investigatorsfrom Ponroy Laboratories had described an experiment in which micelacking essential fatty acids, i.e. linoleic acid (18:2 n-6) andα-linolenic acid (18:3 n-3), which serve as the sole sources forLC-PUFA, were fed cerebral phospholipids and the quantity ofphospholipids in each part of the brain measured. These phospholipidswere found in the cytoplasm, in the synapses, and in other parts of thebrain [Carrie et al., (2000) J. Lipid Res. 41, 465-472].

The utilization of phospholipids enriched with PUFA holds many potentialadvantages from a clinical point of view. The phospholipid may deliverthe essential fatty acid to specific organs or body parts, such as thebrain, and assist in the incorporation of these fatty acids inmembranes. Other advantages may arise from the fact that phospholipidsenriched with PUFA will not have odor problems such as found in themajor current nutraceutical source, the fish oils. Furthermore, somepreliminary clinical studies have shown that PUFA incorporated inphospholipids possess superior efficacy than PUFA carried bytriglycerides. [Song et al. (2001) Atherosclerosis, 155, 9-18].

Further studies have shown that the activity of DHA-rich phospholipidwas different from that of DHA-rich triacylglycerol in spontaneouslyhypertensive rats [Irukayama-Tomobe et al. (2001) Journal of OleoScience, 50(12), 945-950]. Spontaneously hypersensitive rats (SHR) werefed test lipid diets for six weeks, which contained 30%-docosahexaenoicacid (DHA) phospholipid (DHA-PL) extracted from fish roe or 30%-DHA fishoil (DHA-TG). The control diet contained corn oil in the presence oftest lipids. After feeding, blood pressure in the DHA-TG and DHA-PL dietgroups was found significantly lower compared to the control. Serumfatty acid content of dihomo-linoleic acid (DHLnA) and Arachidonic acid(AA) of the DHA-PL diet group was significantly less than the control orDHA-TG diet group. Serum triacylglycerol, phospholipid and totalcholesterol in the DHA-TG and DHA-PL diet groups were significantly lessthan in the control. Liver total cholesterol in DHA-PL was twice that inthe DHA-TG diet group and control. The mechanism for cholesterol removalfrom blood by DH-PL would thus appear to differ from that by DHA-TG.Serum lipid peroxide (LPO) in the DHA-TG and DHA-PL diet groups wasessentially the same as in the control.

Many PUFA-containing agents suffer from stability and quality problemsdue to the high degree of oxidation of the polyunsaturated fatty acids.These problems require the incorporation of antioxidants as well as theutilization of special measures which attempts to reduce this oxidation.The utilization of phospholipids as carriers of PUFA may result inenhanced stability of such products due to the anti-oxidative propertiesof phospholipids.

It seems that one of the most effective transport mechanism for suchessential fatty acids is the attachment of these groups to phospholipidmolecules. The phospholipids have been shown to pass through theblood-brain barrier and transport the DHA where it is needed.

Organoleptic Concerns

PUFAs are traditionally extracted from coldwater fish. Despite thehealthy image, one of the problems of consumer acceptance has been theresulting strong, fishy taste. To address this, microencapsulated formsof omega-3 have been pioneered in the last 15 years. A further step wasthe development of egg-containing products such as DHA-enrichedmayonnaise and pasta. DHA-enriched yogurts, baked goods and broilerswere also envisaged.

There is no other nutritional product or ingredient that is consideredto be an agent of PUFA delivery. All current commercial products arebased on the fatty acids themselves in an encapsulated form or on foodsenriched with PUFA through special animal/crop feed.

It is therefore an object of the present invention to provide lipidpreparations enriched with omega-3 or omega-6 fatty acids, for usemainly as nutraceuticals and as functional food additives. Thecomposition or said preparation is such that it provides the preparationwith the property of enhancing the bioavailability of PUFAs. Thus uponits consumption, preferably in the form of nutraceuticals, foodadditives or pharmaceutical compositions, the organism may, in the mostefficient way, enjoy the benefits provided by said preparation, as willbe described in detail below.

This and other objects of the invention will become apparent as thedescription proceeds.

In a first aspect the present invention provides a lipid preparation,wherein said lipid is selected from glycerophospholipids and theirsalts, conjugates, and derivatives and any mixture thereof, andpoly-unsaturated fatty acid (PUFA) acyl groups, particularly long-chainpoly-unsaturated fatty acid (LC-PUFA) acyl groups, preferably omega-3and/or omega-6 acyl groups, at a concentration of least 5% (w/w) oftotal fatty acids content of said preparation, preferably more than 10%(w/w), more preferably 20-50% (w/w), wherein said PUFA is covalentlybound to said lipid.

Said lipid may be a naturally occurring lipid, or a synthetic lipid.Preferably, said lipid is a glycerophospholipid in which at least someof the sn-1 or sn-2 groups of the glycerol backbone are substituted withsaid poly-unsaturated fatty acid (PUFA) acyl groups.

In one particular embodiment, said lipid is a glycerophosphlipid offormula I:

wherein R″ represents a moiety selected from serine (PS), choline (PC),ethanolamine (PE), inositol (PI), glycerol (PG) and hydrogen(phosphatidic acid—PA), and R and R′, which may be identical ordifferent, independently represent hydrogen or an acyl group, whereinsaid acyl group is selected from saturated, mono-unsaturated orpolyunsaturated acyl groups (PUFA), particularly long-chainpoly-unsaturated fatty acids (LC-PUFA), more preferably omega-3 and/oromega-6 acyl groups, and salts thereof, with the proviso that R and R′cannot simultaneously represent hydrogen, and wherein saidpolyunsaturated acyl groups comprise at least 5% (w/w) of total lipidfatty acids, preferably more than 10% (w/w), and particularly 20-50%(w/w).

In one more particular embodiment of said preparation, R representshydrogen and R′ represents an acyl group. Alternatively, R′ representshydrogen and R represents an acyl group.

Considering these latter embodiments, when said acyl group is preferablyan omega-3 acyl group, it may be an eicosapentaenoyl (EPA), adocosahexaenoyl (DHA) group, or linolenic omega-3 group. And, when saidacyl group is preferably an omega-6 acyl group, it may be anarachidonoyl (ARA) group, or a linoleic omega-6 group. A furtherpossibility is that said acyl group may be a linolenoyl (18:3) group.

In a yet further embodiment of the preparation of the invention, R″ maybe any one of serine, choline, ethanolamine, inositol or glycerol.

In a further particular embodiment, the identity and content of R and R′are predetermined.

The preparation of the invention which comprises the compound of formulaI in which R″ is serine, mimics the composition of human brain PS.

Nonetheless, the invention also refers to preparations comprising thecompound of formula I in which R″ is serine, which are different fromhuman brain PS, but still have an improved bioactivity, particularly ascompared to soybean-PS. This improved bioactivity results in beneficialeffects on both the learning and working memory in elderly population,in particularly in cholinergic impaired conditions like Alzheimer'sdisease.

The invention also relates to preparation PS preparation which mimicsthe human brain PS, is effective at lower dosage (2-3 fold) compared tosoybean-PS, while having similar or improved bioactivity compared tosoybean-PS.

The PS may be of plant, animal or microorganism source, and is enrichedwith PS of formula I, wherein R″ represents a serine moiety.

The preparation of the invention may be further enriched with PS offormula I, characterized in having reduced or absent of fish-relatedorganoleptic effects. Such preparation may be particularly suitable forincorporation into chocolate-containing or dairy-based food articles(including concentrated milk).

The preparation of the invention may be used in the improvement andtreatment of cognitive and mental conditions and disorders as well asthe maintenance of normal functions of brain-related systems andprocesses, preferably ADRD, aging, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis (MS), dyslexia, depression, learningcapabilities, intensity of brain waves, stress, anxiety, mental andpsychiatric disorders, concentration and attention, mood, brain glucoseutilization, general cognitive and mental well being, neurologicaldisorders and hormonal disorders.

The preparation of the invention is particularly useful in enhancing thebioavailability of omega-3 and omega-6 fatty acids.

The preparation of the invention may be used in combined improvement ofcognitive and mental functions together with improvement of additionalhealth disorders or conditions. Such additional health disorders orconditions may be at least high blood cholesterol levels, hightriglycerides levels, high blood fibrinogen levels, HDL/LDL ratio,diabetes, metabolic syndrome, menopausal or post-menopausal conditions,hormone related disorders, vision disorders, inflammatory disorders,immune disorders, liver diseases, chronic hepatitis, steatosis,phospholipid deficiency, lipid peroxidation, dysrhythmia of cellregeneration, destabilization of cell membranes, coronary arterydisease, high blood pressure, cancer, hypertension, aging, kidneydisease, skin diseases, edema, gastrointestinal diseases, peripheralvascular system diseases, allergies, neurodegenerative and psychiatricdiseases.

The preparation of the invention may also be used in the reductionand/or prevention of serum oxidative stress leading to atherosclerosis,cardiovascular disorders and/or coronary heart disease.

The invention further relates to nutraceutical compositions comprising alipid preparation in accordance with the invention. The nutraceuticalcomposition may be in the form of softgel capsules, tablets, syrups, orany other common dietary supplement delivery system.

Still further, the invention relates to functional food articlecomprising the lipid preparation of the invention. Such functional foodarticle may be selected from dairy products, dairy drinks, ice-creams,bakery products, confectionery products, biscuits, soy products, pastryand bread, sauces, condiments, oils and fats, margarines, spreads,cereals, drinks and shakes, oils and fats, infant formulas, infant foods(biscuits, mashed vegetables and fruits, cereals), bars, snacks, candiesand chocolate products.

In yet a further aspect, the invention relates to pharmaceuticalcompositions comprising the lipid preparation of the invention, andoptionally further comprising at least one pharmaceutically acceptableadditive, diluent or excipient. The pharmaceutical composition of theinvention may further optionally comprise at least one pharmaceuticallyactive agent.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWING

FIG. 1A-D: Performance of rats in acquisition of the spatial Morris mazetask.

Latency time to platform in the three days of acquisition (2 sessionsper day) of aged rats supplemented for three months with varioussupplements as detailed below was analyzed using video camera, with(open squares) or without (closed circuits) pretreatment of 1 mg/kg ofscopolamine.

FIG. 1A: Rats supplemented with MCT, P<0.007.

FIG. 1B: Rats supplemented with PS- ω3 P<0.07.

FIG. 1C: Rats supplemented with SB-PS, P<0.02.

FIG. 1D: Rats supplemented with LC-PUFA, P<0.03.

Values represent mean ±S.E.M of four to five rats per supplement.

Abbreviations: Lat. T., latency time; sec., seconds.

FIG. 2. Performance of scopolamine-treated rats in the Morris water mazetask in the spatial probe test.

This graph represents percentage of time (T.) that aged rats,supplemented for three months with MCT (open bars), PS- ω3 (solid bars),SB-PS (dotted bars) or LC-PUFA (striped bars), spent in different areasafter the platform being removed, was analyzed using video camera,following pre-treatment of 1 mg/kg of scopolamine. Values represent mean±S.E.M of four to five rats per supplement. Significance compared tocontrol group (MCT) * P<0.02 and ** P<0.08

FIG. 3A-D: Performance of scopolamine-induced rats in locating theplatform after its reposition.

Latency time to platform on the fifth day of the water maze test, inwhich the platform was repositioned between the sessions, in aged ratssupplemented for three months with different supplements as specifiedbelow, was analyzed using video camera, with (open squares) or without(closed circuits) pretreatment of 1 mg/kg of scopolamine.

FIG. 3A: Rats supplemented with MCT.

FIG. 3B: Rats supplemented with PS- ω3.

FIG. 3C: Rats supplemented with SB-PS.

FIG. 3D: Rats supplemented with LC-PUFA.

Values represent mean ±S.E.M of four to five rats per supplement.

Abbreviations: Lat. T., latency time; sec., seconds; tr., trials.

FIG. 4A-8: Phospholipid levels in rat tissues as measured using ³¹P-NMR.

Lipids were extracted from tissues of aged rats that were supplementedfor three months with MCT (open bars), PS- ω3 (solid bars), SB-PS(dotted bars) or LC-PUFA (striped bars). Phospholipids levels wereanalyzed using a ³¹P-NMR machine and the relative levels ofphosphatidylcholine of the different treatments are presented.

FIG. 4A: Analysis of lipids extracted from the liver.

FIG. 4B: Analysis of lipids extracted from the brain (cortex region).

Values represent mean ±S.D. of four to five rat tissues per supplement.Significance compared to control group (MCT)*P<0.05 and **P<0.1.

Abbreviations: Tot. PI., total phospholipids.

FIG. 5: Parental scores of ADHD children according to behavioral ratingscales.

The graph represents percentage of ADHD children that demonstratedimprovement or lack of improvement in a parental view following twomonths of supplementation with canola oil (open bars), DHA (solid bars)or PS- ω3 (hatched bars). Rating includes remarks regarding behavioraltendencies at home, at school, with siblings or peers and teachersfeedback. Values represent percentage of twenty to twenty-five ADHDchildren scores per supplement. Note that twelve parents decline torespond to the questioner and six children did not complete thesupplementation period due to poor taste or severe discipline problems(mostly the control group).

Abbreviations: Improv., improvement; Marg. Improve., marginalimprovement; n.c., no change; Deter., deterioration.

FIG. 6: Effect of PC-DHA on the serum oxidative stress.

Apo E° mice were fed for 10 weeks with placebo (open bars) or PC-DHA(solid bars). Serum lipid peroxide (Ser. per.) levels were measuredusing a spectrophotometric assay. Values represent mean ±S.D. of 5 miceper treatment.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In a first aspect the present invention provides a lipid preparation,wherein said lipid is a glycerophospholipid, a salt, conjugate, andderivative thereof, and any mixture thereof, and poly-unsaturated fattyacid (PUFA) acyl groups, particularly long-chain poly-unsaturated fattyacid (LC-PUFA) acyl groups, preferably omega-3 and/or omega-6 acylgroups, at a concentration of least 5% (w/w) of total fatty acidscontent of said preparation, preferably more than 10% (w/w), morepreferably 20-50% (w/w), wherein said PUPA is covalently bound to saidglycerophospholipid.

Said lipid may be a naturally occurring lipid, or a synthetic lipid.

Preferably, said lipid is a glycerophospholipid in which at least someof the sn-1 or sn-2 groups of the glycerol backbone are substituted withsaid poly-unsaturated fatty acid (PUFA) acyl groups.

In one particular embodiment, said lipid is a glycerophosphlipid offormula I:

wherein R″ represents a moiety selected from serine (PS), choline (PC),ethanolamine (PE), inositol (PI), glycerol (PG) and hydrogen(phosphatidic acid—PA), and R and R′, which may be identical ordifferent, independently represent hydrogen or an acyl group, whereinsaid acyl group is selected from saturated, mono-unsaturated orpoly-unsaturated acyl groups (PUFA), particularly long-chainpolyunsaturated fatty acids (LC-PUFA), more preferably omega-3 and/oromega-6 acyl groups, and salts thereof, with the proviso that R and R′cannot simultaneously represent hydrogen, and wherein saidpolyunsaturated acyl groups comprise at least 5% (w/w) of total lipidfatty acids, preferably more than 10% (w/w), and particularly 20-50%(w/w).

In one more particular embodiment of said preparation, R representshydrogen and R′ represents an acyl group. Alternatively, R′ representshydrogen and R represents an acyl group.

Considering these latter embodiments, when said acyl group is preferablyan omega-3 acyl group, it may be an eicosapentaenoyl (EPA), adocosahexaenoyl (DHA) group, or linolenic omega-3 group. And, when saidacyl group is preferably an omega-6 acyl group, it may be anarachidonoyl (ARA) group, or a linoleic omega-6 group. A furtherpossibility is that said acyl group may be a linolenoyl (18:3) group.

In a yet further embodiment of the preparation of the invention, R″ maybe any one of serine, choline, ethanolamine, inositol or glycerol.

In a further particular embodiment, the identity and content of R and R′are predetermined.

The preparation of the invention which comprises the compound of formulaI in which R″ is serine, mimics the composition of human brain PS.

Nonetheless, the invention also refers to preparations comprising thecompound of formula I in which R″ is serine, which are different fromhuman brain PS, but still have an improved bioactivity, particularly ascompared to soybean-PS.

Traditionally, PS active ingredients used as dietary supplements wereproduced by the extraction of animal brains, particularly bovine brains.The PS extracted from animal brain tissues, similarly to human brain PS,has a fatty acid composition which is characterized by relatively higherlevels of omega-3 moieties, compared to the levels of omega-3 found inplant phospholipids.PS has the following structure:

Human brain PS is characterized by over 20-30% PS containing omega-3fatty acyls, preferably at the sn-2 position of the glycerol moiety, andmainly DHA or EPA. As mentioned above, phospholipids, and PS inparticular, are responsible for membrane structure and physicalproperties. One of the major physical properties governed byphospholipids is the fluidity of these membranes. Omega-3 fatty acids,DHA and EPA in particular, also have a crucial role in membrane fluidityin light of their unique 3D structure. Therefore, PS with omega-3 fattyacyl moieties, DHA and EPA in particular, has unique bio-functionalitywhich cannot stem from just the basic phospholipid skeleton of thisphospholipid.

Considering the risks involved with prion diseases, particularly bovinespongiform encephalopathy (BSE), as well as other disadvantagesassociated with ingredients obtained from animal sources, PS supplementsare usually prepared using PS originating from soybean lecithin. Thislecithin is enriched, usually enzymatically, with PS. This method ofproduction results in PS with a fatty acid profile of soybeanphospholipids, which is characterized by low level of omega-S fattyacids, and almost no DHA and EPA. This PS active ingredient is alsoknown as soybean-PS.

Although the bio-functionality of soybean-PS in the improvement ofcognitive function has been shown to be similar to that of bovine-PS, itis still different from human brain PS. It is a purpose of the presentinvention to provide a PS ingredient with a predetermined fatty acidcomposition that mimics the fatty acid composition of the human brainPS.

It is a further object of the present invention to provide a PSingredient which, while not identical to naturally occurring brain PS,is characterized by improved functionality, particularly in comparisonwith soybean-PS. This improved PS ingredient has a predetermined fattyacid composition.

The PS ingredient of the present invention is enriched with omega-3fatty acyls, preferably DHA, EPA or linolenic omega-3. Furthermore, thePS of this invention is enriched with omega-3 fatty acyls covalentlybonded to either or both of the sn-1 or sn-2 positions of the glycerolmoiety in the PS backbone.

The present invention is also related and describes other phospholipids,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidyl-inositol (PI), phosphatidylglycerol (PG) and phosphatidicacid (PA), enriched with omega-3 fatty acids, preferably DHA, EPA, orlinolenic acid which are covalently bonded at either or both of the sn-1or sn-2 positions of the glycerol moiety of the phospholipid.Alternatively, the phospholipids of the invention are enriched withomega-6 fatty acids.

When referring to PS in the present description, it should be taken tomean also any other lipid, such as, but not limited to, the polar lipidslisted above.

In a preferred embodiment, the amount of omega-3 (particularly EPA, DHAor linolenic acid) or omega-6 (particularly ARA and linoleic acid) fattyacids in the PS ingredient of the invention is greater than 10% ateither or both of the sn-1 or sn-2 positions, preferably at the sn-2position, preferably over 20% and most preferably above 40%.

As mentioned, the desired omega-3/omega-6 fatty acyls can be bonded atboth or only one of the sn-1 and sn-2 positions.

The fatty acid composition of the PS preparation of this invention canhave a predetermined fatty acid composition similar to or different fromthe fatty acid composition found in normal healthy human brain, providedit has enhanced activity, particularly compared to the activity of plantPS, for example soybean-PS.

The preparation of the omega-3/omega-6-enriched PS preparation of thisinvention can be enzymatic, chemical or by molecular biology methods.Briefly, the PS can be enriched with omega-3 or omega-6 moieties byenzymatic processes, e.g. enrichment of a natural phospholipid/lecithinwith omega-6 fatty acids by enzymatic transesterification/esterificationfollowed by transformation of the head group to serine (using PLDenzymes) to obtain a PS-omega-3/omega-6 conjugate. Another enzymaticpathway is to obtain a lecithin or phospholipid source which isnaturally rich in omega-3 acids, such as krill phospholipids, andtransform their head groups to serine. It is to be noted that the fattyacid composition of the PS obtained by this method has an omega-3composition which is predetermined by the source of choice (fish, krill,algae, etc.). Such methods have been thoroughly described in Applicant'sco-pending PCT Application claiming priority from IL158553.

The PS-omega-3/omega-6 ingredient of the present invention can also beprepared by chemical transesterification/esterification methods thatwill enrich the sn-1 and 2 positions with omega-3 or omega-6 acylresidues. Such methods of preparation of PS-omega-3 and PS-omega-S havebeen described in Applicant's co-pending PCT Application claimingpriority from IL158553.

Alternatively, the PS ingredient of the present invention can beprepared by GMO (genetically modified organisms)/biotechnology methods,for example, providing phospholipids-producing organisms with omega-3 oromega-S fatty acids to obtain phospholipids enriched with omega-3 oromega-6 PS. It may be preferred to use genetically engineered plants ormicroorganisms, to avoid use of animal sources.

The PS of this invention can have the omega-3 or omega-6 fatty acidcomposition of a specific lecithin raw material, relatively rich withomega-3 or omega-6 fatty acids, enriched with PS to yield a PSingredient with elevated omega-3 or omega-6 fatty acids levels, comparedto soybean-PS. Such is the case, for example, when phospholipids fromkrill are used as the starting material, as described above.

In a preferred embodiment the PS enriched with omega-3 or omega-6 can besoybean-PS or any other PS, from plant, animal, for example krill, ormicroorganism source. In a further preferred embodiment the omega-3 oromega-6 enrichment can be performed on a lecithin, which in turn isenriched with PS by transphosphatidylation.

It is the purpose of this invention to provide a novel PS ingredient,enriched with omega-3 fatty acids, resulting in an ingredient withimproved efficacy compared to ingredients containing natural or simplyenriched PS.

The improved PS preparation of this invention exhibits enhanced activityin the improvement and treatment of cognitive and mental conditions anddisorders as well as the maintenance of normal functions of brainrelated systems and processes. These include, but are not limited toADHD, multiple sclerosis (MS), dyslexia, depression, learningcapabilities, intensity of brain waves, stress, mental and psychiatricdisorders, neurological disorders, hormonal disorders, concentration andattention, mood, brain glucose utilization, and general cognitive andmental well being.

The novel lipid preparation of this invention exhibits enhanced activityin the improvement of cognitive functions, as detailed hereunder, overomega-3 or omega-6 lipids per so or soybean-PS. Furthermore, undercertain conditions or for all or specific disorders, the lipidpreparation of the invention is effective at a dosage of less than 100mg/day. This is lower that the current recommended daily dosage ofsoybean-PS (100-300 mg/day) or omega-3 lipids (approx. 1-2 g/day ormore) currently available in the market. Nonetheless, dosages of 100-600mg/day are preferred for enhanced efficacy of the lipid preparation ofthe invention.

An important advantage of the PS preparation of the invention is that itexhibits multifunctional activity. This multi-functionality is exhibitedby improvement in cognitive and mental functions, together withimprovement of other health disorders or conditions.

The enhanced activity of this PS ingredient, as well as itsmulti-functionality, may arise from the unique structure of thisingredient and its influence on the physical and chemical properties ofcell membranes in brain tissues as well as other organs and tissues.

The enhanced activity of this PS ingredient, as well as itsmulti-functionality, may also be attributed to the enhancedbioavailability of the omega-3 fatty acids, due to their incorporationin the PS skeleton. Thus, the omega-3 fatty acids can be delivered tothe brain across the blood-brain barrier, being a part of the PSmolecule, which readily passes this barrier. The PS functions as adelivery platform for the fatty acids bound thereto, to various organsand tissues, thereby enhancing their bioavailability.

The additional health disorders or conditions which are affected by themultifunctional PS preparation of the invention include, but are notlimited to high blood cholesterol levels, high triglycerides levels,high blood fibrinogen levels, HDL/LDL ratio, diabetes, metabolicsyndrome, menopausal or post-menopausal conditions, hormone relateddisorders, vision disorders, inflammatory disorders, immune disorders,liver diseases, chronic hepatitis, steatosis, phospholipid deficiency,lipid peroxidation, dysrhythmia of cell regeneration, destabilization ofcell membranes, coronary artery disease, high blood pressure, cancer,hypertension, aging, kidney disease, skin diseases, edema,gastrointestinal diseases, peripheral vascular system diseases,allergies, airways diseases, neurodegenerative and psychiatric diseases.

The new ingredients of the invention can be delivered and utilized in avariety of products. Such products include dietary supplements,functional foods, pharmaceutical delivery systems, etc.

The preparation of pharmaceutical compositions is well known in the artand has been described in many articles and textbooks, see e.g., GennaroA. R. ed. (1990) Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., and especially pages 1521-1712 therein.

As dietary supplements, the preparations of the invention may be used inthe form of soft gel capsules, tablets, syrups, and other common dietarysupplements delivery systems.

As functional foods, the preparations of the invention can beincorporated and used in a variety of foods, such as dairy products,ice-creams, biscuits, soy products, pastry and bread, sauces,condiments, oils and fats, margarines, spreads, cereals, drinks andshakes, infant formulas, infant foods (biscuits, mashed vegetables andfruits, cereals), bars, snacks, candies, chocolate products.

As pharmaceutical products, the preparations of the invention can bedelivered orally, intravenously, or by any other conventional or specialroute of administration.

The new preparations of the invention may be in the form of fluid oil,powder, granules, wax, paste, oil or aqueous emulsion, and any otherform that will enable its use in the target applications.

Pharmaceutical or nutraceutical formulations comprising the PSpreparation of the invention may include physiologically acceptable freeflowing agents, other additives, excipients, dessicants and diluents,colorants, aroma and taste ingredients, and any ingredients that controlphysical, organoleptic, and other properties, as well as additionalactive ingredients, for example minerals, vitamins, other nutritionaladditives.

The utilization of omega-3 lipids in a variety of applications, andespecially as ingredient of functional foods, is hindered due to theirdistinct fish odor. Thus, another advantage of the omega-3 enrichedphospholipids ingredients of the invention is that they have reducedodor or taste of omega-3 acyl moieties, due to the covalent binding ofthese groups to the PS backbone. This increases the vapor pressure ofthese materials, hence reducing their distinct aroma. Thus, the covalentbinding of the omega-3 fatty acids to the phospholipid backbone,especially PS, alters and improves their taste properties. Moreover, thePS ingredient of the invention also offers enhanced stability to theoxidation sensitive omega-3 fatty acids. Phospholipids in general, andPS in particular, are known to act as anti-oxidants and stabilizers.

These benefits make the lipid preparation of the invention highlybeneficial and important in a variety of applications and especially infunctional foods, where stability, aroma and taste are fundamentalrequirements.

Furthermore, these novel ingredients can be formulated with additionallipids for an even enhanced bio-functionality and efficacy.

The polar lipids derivatives of PUFA, such as the PS-PUFA derivativeshave exhibited high stability as a preparation and additionally inseveral food applications, used in the clinical trials of the presentinvention. The stability of these sensitive compounds is emerging fromthe covalent combination of phospholipids, known in the past to be usedas preservatives and of the un-stable PUFA moieties.

The new ingredients of the invention can be delivered and utilized in avariety of products. Such products include dietary supplements,functional foods, pharmaceutical delivery systems, etc.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Example 1 Methods

Animals and Diet

Male Wistar rats originated from the same colonies were obtained fromHarlen. Fifty rats were randomly divided into five dietary supplementedgroups, in addition to their normal diet: (i) a group fed 0.1 gmedium-chain triglycerides (MCT)/1 ml supplement matrix (MCT group);(ii) a group fed 0.1 g DHA/EPA (20/30% of total fatty acids composition,diluted with MCT to generate 30% (w/w) LC-PUFA compound) triglycerides/1ml supplement matrix (LC-PUFA group); (iii) a group fed 0.1 g soybeanlecithin-derived PS (20% SB-PS w/w)/1 ml supplement matrix (SB-PSgroup); and (iv) a group fed 0.1 g PS- ω3 (20% PS w/w, and total LC-PUFAcomposition of 30%)/1 ml supplement matrix (PS group). The supplementmatrices were stored at −20° C., and fresh portions were fed to the ratsevery day. All supplements were handled so as to minimize oxidation ofthe fatty acids. Rats consumed the diet and water ad libitum. All ratswere housed in a standard environment, in which temperature wasmaintained at 24±0.5° C., and the relative humidity was kept at 65±5%with 12-h periods of light and dark. Body weight was measured at thebeginning and the end of the treatment period.

The PS- ω3 compound used in this study mimics the fatty acidscomposition of the mammalian brain PS, with respect to its DHA content(20%). Generally, in animal cells, the fatty acid composition of PSvaries from tissue to tissue, but does not appear to resemble theprecursor phospholipids, either because of selective utilization ofspecific molecular species for biosynthesis or because or re-modeling ofthe lipid via deacylation-reacylation reactions. In human plasma,1-stearoyl-2-oleoyl and 1-stearoyl-2-arachidonoyl species predominate,but in brain and many others related tissues1-stearoyl-2-docosahexaenoyl species are very abundant [O'Brien et al.(1964) J Lipid Res. 5(3):329-38]. An early work by Yabuuchi et al.[Yabuuchi et al. (1968) J Lipid Res. 9(1):65-7] established that the DHAcontent in bovine gray matter is up to 30% of the total fatty acidscomposition; most of the total amount of DHA was located at the sn-2position (60%). It was the bovine brain PS that Toffano and Brunireported in the early 1980's to be a pharmacologically active compound,which counteracts age-related changes in the central nervous system[Toffano et al. (1980) Pharmacol. Res. Commun. 12:829-845].

Behavioral Testing

Water maze test, which was developed by Morris [Stewart, C A. andMorris, R G. (1993) The water maze. In: Behavioural Neuroscience: APractical Approach. Vol. 1 (Saghal, A., ed.), pp. 107-122. OxfordUniversity Press, New York, N.Y.], uses a circular tank (137 cmdiameter, 35 cm deep) constructed of opaque white plastic. It is filledwith water (21-22° C.) to a depth of 28 cm, and the water is renderedopaque by the addition of soluble, nontoxic white latex paint. In theplace version of the maze, the rat develops a spatial map of theextra-maze cues, which it then uses to locate the platform. Thus thedistance swum to the platform and the time taken in doing so shoulddecrease over testing sessions (days) as the rat learns the location ofthe platform. Moreover, it is expected that if the rat has learned thelocation of the platform in relation to the extra-maze cues, its initialresponse on the probe trial will be to swim directly to the quadrant inwhich it expects to find the platform. Thus the distance swum (and timespent) in the target quadrant should be greater than that in the othertwo quadrants (excluding the start quadrant). The distance swum to theplatform as well as the latency to reach the platform were monitoredusing the video-based tracking system. The behavioral testing wasconducted during the dark cycle, when rats are normally most active.

The pool was located in a test room in which there were many extra-mazespatial cues. On the first three days, the rats were required to locatethe hidden platform (15.5 cm×15.5 cm) situated 1 cm below the surface ofthe water. There were two acquisition testing sessions per day, withfour trials per session. On each trial, the rat was placed, facing thewall, in one of the four quadrants in the tank, and allowed to swim fora maximum of 60 seconds. Once the rat found the platform, it remainedthere for 5 seconds before being returned to the holding cage, which waskept warm on a heating pad. If the rat failed to find the platform inthat time, it was placed on it for 5 seconds before being returned tothe holding cage. Each of the eight trials conducted each day wasstarted from a different quadrant, with the order determinedpseudorandomly (not twice from the same quadrant) and varying from dayto day. The intertrial interval (ITI) was 120 seconds, counted from theend of one trial to the beginning of the next. On fourth day, followedby a session as abovementioned, the platform was removed from the tank,and a probe trial was conducted by placing the rat in the quadrantopposite to that of the platform and then allowing it to swim for 60seconds. The day following the probe trial, the rats were tested with asession in which the maze was set up as previously described, followedby a session in which the platform was repositioned to the center of theopposite quadrant. The latency to find the platform on each trial wasrecorded. Scopolamine (1 mg/Kg) was intraperitoneally (i.p.)administered 30 minutes before the indicated trials.

Lipid Extraction and NMR Analyses

At the end of the behavioral testing, the rats were anesthetized withHalothane and then decapitated. Liver and brain tissues were quicklyremoved and stored (at −80° C.). The lipid fraction of the rat tissueswere extracted using a modified version of the technique described byBligh and Dyer 1959 [Bligh and Dyer, (1959) Can. J. Biochem. Physiol.37, 911-917]. Briefly, 500-700 mg and 300-1200 mg of liver and braintissues, respectively, were homogenized in a solution of CDCl3, methanoland CS-EDTA (1:2:2 v:v:v). The homogenates were further agitated usingultrasonic bath (10 min, 80° C.), followed by additional vigorousshaking (20 min). The relative ratio of the phospholipids in thehomogenates was measured using high-resolution ³¹P-NMR at 121.MHZ usinga 7.06 Tesla General Electric spectrometer.

These homogenates were further analyzed for their fatty acidsdistribution. First, the lipids extracts were desalted by reverse-phasechromatography using an RP-18 column [Williams et al. (1980) J.Neurochem.; 35, 266-269]; diheptadecanoyl phosphatidylcholine was addedas internal standard before the loading on the column. Phospholipidswere separated from neutral lipids, such as cholesterol, on silica gelplates (Merck 60) developed in isohexane: ether: formic acid 80:20:2(v:v:v). The phospholipids spot was visualized by spraying primulinsolution and compared with authentic phospholipids standards.Henicasonoic methyl ester (C21:0) was added as a 2nd internal standardand the phospholipids were converted to methyl esters by mild acidhydrolysis with 1% methanolic H2SO4 overnight at 50° C. The fatty acidsprofile of the different samples was determined by gas-liquidchromatography.

Results

Anti-dementia effects of bovine brain cortex-derived PS (BC-PS) has beendemonstrated by several double-blind, placebo-controlled studies, seereview by [Kidd P. (1996) Alt Med. Rev. 1(2):70-84]. In the past decadeboth BC-PS and soybean lecithin transphosphatidylated PS (SB-PS) wereshown to recover the scopolamine-induced amnesia in rodent, although thefatty acids composition is considerably different between thesecompounds [Zanotti A et al. (1986) Psychopharmacology (Berl).90(2):274-5; Claro F. et al. (1999) Physiol Behav. 67(4):551-4; Sakai M.(1996) Nutr Sci Vitaminol. (Tokyo) 42(1):47-54; Furushiro M et al.(1997) Jpn J Pharmacol. 75(4):447-50]. The means of PS administration inthese studies was predominantly intravenous or intraperitoneal; althoughFurushiro et al. described also oral administration of SB-PS thatantagonized amnesic effects of scopolamine. However, in the latter studythe investigator used a considerable high dose of SB-PS, ranging between60 to 240 mg/Kg.

In the presented study, rat diet was supplemented with theabove-mentioned treatments (diets i, ii, iii, iv and v) for three monthsbefore the maze test was performed. In the acquisition stage (FIG.1A-1D) there is an expected and marked increase in the latency time tofind the platform after the administration of scopolamine (1 mg/Kg) ofall groups. Although the latency curves of MCT and PS- ω3 groups aresimilar, there is a statistically smaller difference in the latencychange, induced by scopolamine, in the PS- ω3 group with respect to thelatency presented by the MCT group (P-value<0.07 Vs. P-value<0.0007,respectively). Similarly, the groups treated with SB-PS or LC-PUFA,demonstrated a reduced effect of scopolamine on their learning curves,with respect to the MCT group (see FIG. 1A-10). Having all groups learnthe task at a similar rate, resembles data presented by Blokland et al.[Blokland et al. (1999) Nutrition 15(10): 778-83], which showed nodifference between PS obtained from different sources and the emptyvehicle, in a water maze test.

What is particular to the present trial is the accelerated rate inlearning the task under the scopolamine sedation. This was notdemonstrated previously [Furushiro et al., (1997) id ibid.; Suzuki etal., (2000) Jpn. J. Pharmacol. 84, 86-8]. Note that in these studies therodent faced a different task (passive avoidance). In Suzuki et al. 2001(J. Nutr. 131: 2951-6) the investigators utilized considerably olderrats (24-25 months old) than the ones tested in the present trial. Thelatency time in the acquisition step was considerably longer for theaged rats compared to the young ones that were tested (eight weeks).Interestingly, although the latency time in the present trial ofnon-sedated rats is somewhat comparable to the younger rats tested bySuzuki et al. [Suzuki et al. (2001) id ibid.], the scopolamine-inducedamnesia latency time in the MCT group resembles the one obtained at thedescribed study for elderly rats. In conclusion, scopolamine induced acomparable long latency time in the control group (MCT). This effect wasaugmented to a different extent by long-term treatment of rats witheither PS or LC-PUFA.

In the probe trial, the rats treated with PS- ω3 showed a distinctivelyhigher tendency than MCT-treated ones (P<0.085) to be present at thezone in which the platform was located during the acquisition of thetask (FIG. 2), indicating that the rats had learned the spatial locationof the platform. Moreover, PS- ω3 treated rats presented a reducedtendency (P<0.08) to swim in the periphery zone, but rather spent in thecentral zone. These latter indications, presented by the PS- ω3 groupare related to a higher adventurous characteristic and could be somewhatcorrelated with the open field behavior trial. Interestingly, inBlokland et al. [Blokland et al. (1999) id ibid.] BC-PS treated micedemonstrated a non-significant but clear tendency to be less adventurousin the open field behavior trial, by spending less time in the centerarea. With respect to the remarkable learning abilities demonstrated bythe rats that were treated with PS- ω3, it is interesting to comparetheir performance in the Morris water maze task in the spatial probetest to the one obtained by the SB-PS treated animals by Suzuki et al[Suzuki et al (2001) id ibid.]. Though the percent of time spent in thequadrant where the platform was located is similar (˜45%), it isremarkable that the dosage in the current study was merely one third ofthe administration levels in Suzuki et al 2001 (20 mg/kg vs. 50 mg/kg,respectively). Indeed, in the present study there was no significantchange in the time that the SB-PS (20 mg/kg) treated rats spent in thisquadrant when compared with the values obtained by the MCT-treated group[FIG. 1C and FIG. 1A, respectively]. In summary, the PS- ω3 treatedgroup learning abilities were markedly higher than the control, in aconsiderably low level of PS administration. In addition, the ratstreated with PS- ω3 were less conservative and more adventurous instudying the maze in the absence of the platform.

Finally, the most prominent and outstanding data obtained in the presentstudy was the response to the repositioning of the platform. All groupspresented a shorter latency in finding the platform at the firstsession, when compared to the one obtained by the MCT-treated group,under scopolamine sedation (FIG. 3A-3D). These data suggest thatLC-PUFA, and more potently PS, can attenuate scopolamine-inducedamnesia, as previously presented by other studies (see selectedreferences above).

Surprisingly, in the second session, there were no differences betweenthe latency in finding the platform after its repositioning in allgroups but the PS- ω3 treated group. In fact, it seemed that in alltreatments but the PS- ω3 there was no learning process of the positionof the platform. The PS- ω3 group presented a remarkably differentbehavior; it seemed that there was no lag in the learning of therepositioned platform in the rat treated with this anti-muscarinic drug.The ability of the PS- ω3 treated group to locate the platform after ithad been repositioned seemed to be contradictory with the resultobtained earlier in the spatial probe test (FIG. 2), where these ratsshowed preference for the third quadrant. Pearce and colleagues [Pearceet al. (1998) Nature 396: 75-77] attempted to resolve this discrepancy,by describing two means for memorizing a specific spatial location. Oneis to use a cognitive map that encodes information about the geometricrelationship between the object and several land marks (the cognitivemap method) and the other is the use of heading vectors that specify thedirection and distance from a single landmark to the object (the headingvector method). In the present test, the rats could locate the platformfrom the above-mentioned cues and/or from the distance and directionwith respect to the walls. In the acquisition and the spatial probetest, both methods contributed to the score of finding the platform.However, in the repositioning test, the cognitive abilities which arerelated to the heading vector method and the short-term memory (workingmemory), made the difference. The heading vector method, because thedistance from the wall was not effected by the repositioning (just thequadrant), and the working memory due to the benefits in memorizing theareas already explored that enable an effective search in the pool.

It has been previously reported that the mechanism by which PSattenuates the scopolamine effect could be attributed not only to abeneficial effect on the cholinergic circuitry, but PS could also havean effect on the serotonergic neuronal system [Furushiro et al. (1997)id ibid.]. It appears that the presented data could be the result ofmore than one neuronal system alteration, possibly the dopaminergic. Inan earlier study [Drago et al. (1981) Neurobiol Aging, 2(3):209-13], itwas suggested that the alteration in the obtained behavioral changesbetween BC-PS treated aged rats to their control could be attributed notonly to the modifications in cholinergic and serotonergic transmission,as described above, but also through affecting the catecholaminergic(like dopamine) system. In this study the facilitated acquisition ofactive avoidance behavior as studied in shuttle-box and pole jumpingtest situations, and the retention of active and passive avoidanceresponses were improved in the PS-treated rats. Tsakiris [Tsakiris, S.(1984) Z Naturforsch [C], 39(11-12):1196-8] reported on an indirecteffect of PS on the dopamine related adenylyl cyclase, through membranefluidity mechanism. Interestingly, it has also been reported [Chalon, etal. (1998) J Nutr.; 128(12):2512-9] that enriched diet with high levelof (n-3) PUFA could result in an effect on the cortical dopaminergicfunction. It is conceivable that the existence of LC-PUFA on thebackbone of the phospholipids was highly beneficial in terms of such amulti-neurotransmitter mechanism.

The biochemical analyses of the present results in liver tissues (FIG.4A) shows that in rats supplemented with PS for three months (SB-PS andPS- ω3) there was a notable increase in the levels of the primerphospholipids, i.e. phosphatidylcholine (PC). These data is consistentwith early observations regarding the liver and its major role in thephospholipids uptake and the primary metabolism of most fatty acids.Wijendran and colleagues [Wijendran et al. (2002) Pediatr. Res.51:265-272] described a study in which baboons were fed labeled LC-PUFAon the backbone of PC and triglycerides, and demonstrated that thelevels of incorporation of LC-PUFA on a phospholipid backbone to theliver was higher than the extent of incorporation of LC-PUFA on thetriglycerides backbone. In addition, PS levels of rats fed with PS- ω3were elevated in cortex tissues analyses of phospholipids distribution(FIG. 4B), comparing with MCT. Interestingly, the phospholipids fattyacids profile of these cortices (Table 1) demonstrate a marked elevationin the DHA content of the rats fed with PS- ω3 (P=0.007). Similarelevation was noted for LC-PUFA fed rats, however to a reduced extentcompared with PS- ω3 treatment (14.6 versus 17.5, respectively PS- ω3)and MCT (14.6 versus 12.3, respectively P=0.02). This difference in theDHA levels between the two omega-3 groups might suggest enhancedbioavailability of DHA when it is esterified to the backbone ofphospholipids rather than to triglycerides. Similar conclusions weredrawn by Lemaitre-Delaunay and colleagues [Lemaitre-Delaunay et al.(1999) J. Lipid Res.; 40:1867-1874], when they had study the kineticsand metabolic fate of labeled DHA on triglycerides versus its enrichmentin lysophsphaytidylcholine, and by Wijendran et al. [Wijendran et al.(2002) id ibid.] in the above-mentioned baboons study.

Interestingly, this increase in DHA content in the cortices of both PS-ω3 and LC-PUFA fed rats is accompanied with a statistically significantdecrease in the levels of oleic acids and to somewhat lower extent oflinoleic acid (Table 1) in the phospholipids fraction. Similar changesin the ratios of the fatty acids profile was demonstrated by others, byfeeding rodents with dietary fats enriched with LC-PUFA [for example:Yamamoto et al. (1987) J. Lipid Res. 28: 144-151]. The SB-PS groupshowed a very similar profile to the MCT group.

In sum, the improved performance in the Morris water maze test of thePS- ω3 treated rats under scopolamine sedation strongly supports thepotency of PS- ω3 as an anti-dementia and age-associated memoryimpairment effects. This cognitive enhancement is further supported bythe biochemical evidence of the elevated phospholipids levels in theliver and brain tissues (FIG. 4A-4B), and with elevated levels of DHAattached to the phospholipids from the cortex of the PS- ω3 fed rats.

Table 1 summarizes the effect of dietary LC-PUFA from different sourceson the fatty acids profile in cerebral phospholipids from elderly Wistarrats. Fatty acids from the purified phospholipids fraction were analyzedby gas-liquid chromatography. The major fatty acids are expressed as %of total fatty acids in the phospholipids. Values represent mean ±S.D.of four different rats per treatment. Statistical significant betweendifferent supplements and MCT group is presented as followed: * P<0.05;** P<0.01. TABLE 1 Fatty acids MCT LC-PUFA SB-PS PS- ω3 C16:0 12.9 ±1.4  14.6 ± 4.7  13.7 ± 4.7  13.6 ± 4.4  C16:1 1.0 ± 0.7 1.0 ± 0.3 1.5 ±0.4 1.5 ± 0.8 C18:0 17.9 ± 1.0  20.1 ± 1.3* 17.2 ± 2.8  18.0 ± 5.5 C18:1 36.5 ± 1.8  32.0 ± 2.8* 37.0 ± 6.8  30.7 ± 4.1* (n-9) C18:1 3.7 ±0.5  4.3 ± 0.2* 4.0 ± 0.3 4.8 ± 1.5 (n-7) C18:2 7.2 ± 0.7  4.5 ± 0.6**7.1 ± 2.6 5.1 ± 2.7 C20:1 2.5 ± 0.5 2.9 ± 0.8 2.1 ± 0.4 2.3 ± 0.3 C22:612.3 ± 1.7  14.6 ± 0.6* 12.4 ± 3.2   17.5 ± 2.4** C24:1 3.4 ± 1.0 3.3 ±1.3 2.8 ± 0.9  2.0 ± 1.2* rest 2.7 ± 0.1 2.8 ± 0.4 2.1 ± 0.9 4.5 ± 3.0

Example 2 PS-omega-3 in the Treatment of ADHD Children

Attention-deficit/hyperactivity disorder (ADHD) encompasses a broadconstellation of behavioural and learning problems and its definitionand diagnosis remain controversial [Kamper (2001) J. Pediatr. 139:173-4;Richardson et al. (2000) Prostaglandins Leukot. Essent. Fatty Acids,63(1-2):79-87]. The etiology of ADHD is acknowledged to be both complexand multi-factorial. Traditionally, ADHD is the diagnosis used todescribe children who are inattentive, impulsive, and/or hyperactive.Roughly 20-25% of children with ADHD show one or more specific learningdisabilities in math, reading, or spelling [Barkley, R. A. (1990)Attention-deficit hyperactivity disorder a handbook for diagnosis andtreatment. New York: Guilford Press]. Children with ADHD often havetrouble performing academically and paying attention, and may bedisorganized, have poor self-discipline, and have low self-esteem. Aconservative estimate is that 3-5% of the school-age population has ADHD[American Psychiatric Association. Diagnostic and statistical manual ofmental disorders. 4th ed. (DSM-IV) Washington, D.C.: AmericanPsychiatric Association, 1994]. Treatments for ADHD include behaviortherapy and medications, mainly methylphenidate (Ritalin™).Psychostimulant drugs and antidepressants are often used to calmchildren with ADHD, with an effectiveness rate of ˜75% (Swanson et al.Except Child 1993; 60:154-61). The advantages of using these medicationsinclude rapid response, ease of use, effectiveness, and relative safety.Disadvantages include possible side effects, including decreasedappetite and growth, insomnia, increased irritability, and reboundhyperactivity when the drug wears off [Ahmann et al. (1993) Pediatrics;91:1101-6]. Moreover, these medications do not address the underlyingcauses of ADHD. Thus, studies to elucidate the potential contributors tothe behavior problems in ADHD may lead to more effective treatmentstrategies for some children.

Omega-3 fatty acids are specifically implicated in maintaining centralnervous system function. Deficiency of n-3 fatty acids in rats andmonkeys has been associated with behavioral, sensory, and neurologicaldysfunction [Yehuda et al. (1993) Proc. Nat. Acad. Sci. USA; 90:10345-9;Reisbick et al. (1994) Physiol. Behav. 55.231-9; Enslen et al. (1991)Lipids; 26:203-8]. Several studies have focused on essential fatty acidmetabolism in children with ADHD [Colquhoun et al. (1981) MedHypotheses; 7:673-679]. Children with hyperactivity have been reportedto be more thirsty than normal children and have symptoms of eczema,asthma, and other allergies [Mitchell et al. (1987) Clin. Pediatr.;26:406-11]. For example, in a cross-sectional study in 6-12-y-old boysrecruited from central Indiana, it was showed that 53 subjects with ADHDhad significantly lower proportions of key fatty acids in the plasmapolar lipids [arachidonic acid (AA; 20:4n-6), eicosapentaenoic acid(EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3)] and in redblood cell total lipids (20:4n-6 and 22:4n-6) than did 43 controlsubjects [Stevens et al. (1995) Am. J. Clin. Nutr; 62:761-8]. However,recent publications [Hirayama et al (2004) Eur. J. Clin. Nutr.;58(3):467-73; Voigt et al (2001) J Pediatr.; 139(2):189-96] thatinvestigated whether DHA supplementation would result with amelioratethe symptoms in ADHD children, suggested that careful attention shouldbe paid as to which fatty acid(s) is used. In these studies DHAsupplementation had demonstrated only marginal if any beneficialeffects.

Recently, it has been suggested that one of the possible solutions tothe nutrient deficiencies which are common in ADHD, could be PSsupplementation [Kidd (2000) Altern Med Rev.; 5(5):402-28].

Method

Subjects and Diet

Ninety 8-to-13-year old children diagnosed according to the DSM-IV asADHD, were assigned randomly, in a double-blind fashion to receive PS-ω3 (300 mg/d; containing total 450 mg/d DHA/EPA), 450 mg/d DHA/EPA orcanola oil (30 per group) for two months, while not taking stimulantmedication or other supplements. Characterizing the subject as ADHDincluded a score lower than −1.8 in the Test of Variables of Attention.

Data Analysis

At the conclusion of the trial, ADHD children were scored according toparental behavioural rating scales (Connors' Rating scale).

Results and Discussion

Use of complementary therapies is particularly common among patientswith chronic, incurable, or frequently relapsing conditions. Forexample, use of complementary and alternative medical therapies (CAM) iscommon in children with cancer, asthma, and cystic fibrosis. Parents orsubjects who seek CAM typically do so because such therapies are moreconsistent with their values, are more empowering, and are perceived asmore natural and less risky than conventional treatments. The majorityof these patients do not abandon mainstream therapies but use herbs andother forms of CAM as adjunctive treatments. Only a minority (<40%) talkwith their pediatricians about their use of CAM. Because of the stigmaand side effects that accompany use of stimulant medications, manyfamilies turn to CAM to treat ADHD. Typically, only 70% of childrenrespond to stimulants such as Ritalin™, and of those who do,approximately half report side effects from their medications. In anAustralian survey of 290 families seen at a multidisciplinary referralcenter for ADHD, 64% had tried at least one “other therapy,” mostcommonly dietary restriction, multivitamin supplementation, andoccupational therapy [Stubberfield et al. (1999) J Paediatr Child Heath;35:450-3].

In the presented study the different supplementation was formulated intoa popular chocolate paste (see below). Using this matrix enable theparents to administer the treatments in a non-conventional form to theirchildren and provided a reduced organoleptic effect characteristic ofthe marine-derived compounds (see below).

The parental rating survey, at the end of the treatment period, measuredthe attention deficit, hyperactivity and impulsivity of the children, aswell as the aggression as assessed by parents, teachers, siblings andpeers. The results indicate a distinctively large placebo effect. Thiseffect is somewhat reduced if the placebo-treated ADHD children thatfailed to complete the study due to severe behavioral deterioration aretaken into consideration. It seemed that most of these children insistedon reassigning for Ritalin™ administration. However, the present dataalso clearly demonstrate PS- ω3 as a potent agent. All in all, ˜70% ofthe parents of the PS- ω3 treated ADHD children indicated someimprovement in the behavioural score of their children, whereas 50% ofthese parents provided clear indications for multiple beneficial effectof the supplement on their children behavior. This prominent effect is2.2-fold higher than the improvement obtained by placebo (˜30%).Comparison of the parental scoring of LC-PUFA on ADHD children behaviorwith the parallel rating that followed three months of PS- ω3administration, point at the latter to have a higher score. While bothcompounds demonstrated similar extent of marginal improvement, PS- ω3had a marked higher rate of substantial improvement (47% versus 35%,respectively) with the lowest rats of lack or deteriorating effects (21%& 11% versus 26% and 17%, respectively). These effects of PS- ω3supplementation could be attributed to both enhanced bioavailability ofomega-3 fatty acids and through PS well documented effects on mood,stress and anxiety.

Example 3 Effect of PC-DHA Consumption in ApoE° Mice

Methods

Animal Diet

Apolipoprotein E deficient (ApoE°) mice [Hayek T. et al. (1994) Biochem.Biophys. Res. Commun. 201:1567-1574] at 8 weeks of age, were assignedrandomly (5 mice each) to LC-PUFA enriched lecithin (30% omega-3 oftotal fatty acids composition; PC-DHA group) or placebo. The mice werefed, besides the regular chow diet, once every three days with either 25μl PC-DHA or PBS, via oral gavage, during 10 weeks.

Each mouse consumed approximately 5 mL of water/day, and 5 g ofchow/day.

Serum Lipids Peroxidation

Serum was diluted 1:4 in PBS. Serum susceptibility to oxidation wasdetermined by incubating serum) sample with 110 mM of the free radicalgenerating compound, 2′-2′-azobis 2′-amidinopropane hydrochloride(AAPH), which is an aqueous soluble azo compound that thermallydecomposes to produce peroxyl radicals at a constant rate. The formationof thiobarbituric reactive substances (TBARS) and of lipid peroxides wasmeasured and compared to serum that was incubated under similarconditions, but without AAPH.

Results and Discussion:

ApoE° mice are widely used as an animal model for atherosclerosis asthey develop severe hypercholesterolemia and atherosclerotic lesions ona chow diet. Moreover, accelerated atherosclerosis is associated withincreased lipid peroxidation of plasma lipoproteins and arterial cellsin these mice [Hayek T. et al. (1994) id ibid., Keidar S. (1998) LifeSci. 63:1-11].

FIG. 6 shows how prolonged PC-DHA consumption by ApoE° mice resulted ina clear tendency (P<0.10) to reduce the serum susceptibility toAAPH-induced oxidation by 16% (in comparison to placebo).

Organoleptic Issues

The utilization of omega-3 lipids in a variety of applications, andespecially as ingredient of functional foods, is hindered due to theirdistinct fish odor. Thus, another advantage of the omega-3 enrichedphospholipids ingredients of the invention is that they have reducedodor or taste of omega-3 acyl moieties, due to the covalent binding ofthese groups to the PS backbone. This increases the vapor pressure ofthese materials, hence reducing their distinct aroma. Thus, the covalentbinding of the omega-S fatty acids to the phospholipid backbone,especially PS, alters and improves their taste properties. Moreover, thePS ingredient of the invention also offers enhanced stability to theoxidation sensitive omega-3 fatty acids. Phospholipids in general, andPS in particular, are known to act as anti-oxidants and stabilizers.

These benefits make this novel phospholipids' preparation of theinvention highly beneficial and important in a variety of applicationsand especially in functional foods, where stability, aroma and taste arefundamental requirements.

Furthermore, these novel ingredients can be formulated with additionallipids for an even enhanced bio-functionality and efficacy.

The starting compound used for the above-mentioned clinical trial inADHD patients, was LC-PUFA enriched PS mixed with fish oil. Originally,this product and the control fish oil were formulated in food productslike energy bars; however the responses from expert panels werecategorically devastating, pointing at severe organoleptic problems. Inorder to overcome this taste barrier the PS- ω3 product of the inventionwas de-oiled. The end-product of this process was a paste that whenreformulated with either inert or dominant-organoleptic saturated fatscould be easily formulated in chocolate bars, chocolate spread,chocolate coated cornflakes, low-fat dairy products or concentratedmilk. Each one of these formulations had an evidently reducedorganoleptic objection from both the expert panels and the trialvolunteers.

The polar lipids derivatives of PUFA, such as the PS-PUFA derivativeshave exhibited high stability as a preparation and additionally inseveral food applications, used in the clinical trials of thisinvention. This stability, of these sensitive compounds is emerging fromthe covalent combination of phospholipids, known in the past to be usedas preservatives and of the un-stable PUFA moieties.

The stability of a commercially prepared fish oil (omega-3 fatty acid)for laboratory rodent diet [Lytle et al. (1992) Nutr Cancer;17(2):187-94] or as an enrichment in spreadable fats [Kolanowski et al.(2001) Int J Food Sci Nutr.; 52(6):469-76] was addressed by severalstudies as the public awareness towards the beneficial effects ofLC-PUFA increased. A major effort was directed at maintaining theoxidative stability of the fish oil, as these fatty acids are subject torapid and/or extensive oxidation and other chemical changes by exposureto air, light, or heat during processing or when stored for variouslengths of time. The common solution presented in these studies wassupplementation the fish oil matrix with antioxidants like butylatedhydroxytoluene, butylated hydroxyquinone and alpha-tocopherol, oralternatively, dilution of concentrated fish oil to a limit of 1% in asaturated fats matrix. However, Song and colleagues [Song et al. (1997)Biosci Biotechnol Biochem.; 61(12):2085-8] had already evaluated theperoxidative stability of DHA-containing oils the form of phospholipids,triglycerides, and ethyl esters in the dark at 25° C. in a bulk phaseduring 10 weeks storage. They had shown that DHA-containing oil in theform of phospholipids was more resistant to the oxidative degradation ofDHA than that in the form of triglycerides and ethyl esters in a bulkphase.

The abovementioned PS- ω3 containing products utilized for the clinicalstudies were tested for their shelf-life and stability in roomtemperature. The enriched PS- ω3 formulated in condensed milk (1 gproduct per 10 ml milk) was analyzed by ³¹P-NMR for stability in cyclesof freeze-thawing for a week, and was found to be stable. In the secondphase, PS- ω3 in a chocolate paste matrix (0.75 g product per 20 gchocolate spread) was tested for stability after two weeks storage inroom temperature. This formulation also presented a stable percentage ofPS, in ³¹P-NMR analysis. In conclusion, we had been able to establishthat ω-S containing phospholipids are highly stable in room temperature,as well as in freezing-thawing cycles, as oppose to ω-3 containingtriglycerides known to rapidly decay after antioxidant consumption.

1. A method for the maintenance, improvement and treatment of behavioraland learning disorder comprising: administering to a subject in needthereof a lipid preparation, wherein said lipid is a glycerophospholipidof formula I:

wherein R″ represents a serine moiety, and R and R′, which may beidentical or different, independently represent hydrogen or an acylgroup, wherein said acyl group is selected from saturated,mono-unsaturated or poly-unsaturated fatty acids (PUFA), particularlylong-chain poly-unsaturated fatty acids (LC-PUFA), more preferablyomega-3 and/or omega-6 acyl groups, and salts thereof, with the provisothat R and R′ cannot simultaneously represent hydrogen, and wherein saidLC-PUFA constitute at least 5% (w/w) of total fatty acids content ofsaid preparation, preferably more than 10% (w/w), and particularly20-50% (w/w) wherein said lipid preparation is a pharmaceutical ornutraceutical composition, or functional food, wherein said subject is achild and wherein said behavioral and learning disorder is selected fromthe group consisting of Attention Deficit Hyperactivity Disorder (ADHD),dyslexia, depression, memory impairment, intensity of brain waves, brainglucose utilization, stress, anxiety, mental and psychiatric disorders,concentration and attention, mood, general cognitive and mental wellbeing, neurological disorders and hormonal disorders.
 2. The methodaccording to claim 1, wherein said cognitive and mental condition isADHD.
 3. The method according to claim 1, wherein said lipid is anaturally occurring lipid, or a synthetic lipid.
 4. The method accordingto claim 1, wherein R represents hydrogen and R′ represents an acylgroup.
 5. The method according to claim 1, wherein R′ representshydrogen and R represents an acyl group.
 6. The method according toclaim 1, wherein said lipid has an omega-3 or omega-6 fatty acidcomposition of lecithin from a marine source.
 7. The method according toclaim 6, wherein said marine source is selected from fish, krill andalgae.
 8. The method according to claim 1, wherein said omega-3 acylgroup is selected from the group consisting of eicosapentaenoyl (EPA),docosahexaenoyl (DHA) and omega-3 alpha-linolenoyl group.
 9. The methodaccording to claim 1, wherein said omega-6 acyl group is selected fromthe group consisting of arachidonoyl (ARA), omega-6 linoleoyl andomega-6 gamma linolenoyl group.
 10. The method according to claim 1,wherein R and R′ are different and selected from EPA and DHA.
 11. Themethod according to claim 1, wherein said PUFA has increasedbioavailability to the organism when comparing to a compositioncomprising PUFA alone or not esterified to said phospholipid backbone.12. The method according to claim 1, wherein said glycerophospholipidmimics the fatty acid composition and/or fatty acid profile of humanbrain phosphatidylserine (PS) and/or mammalian brain PS.
 13. The methodaccording to claim 1, wherein said glycerophospholipid is different fromhuman brain PS and has improved bioactivity compared to soybean-PS. 14.The method according to claim 1, wherein said glycerophospholipid isderived from any one of plant, animal or microorganism source.
 15. Themethod according to claim 1, wherein said glycerophospholipid issynthetic.
 16. The method according to claim 15, wherein saidglycerophospholipid is prepared by enzymatic transphosphatidylation of alipid source, preferably any one of plant, animal or microorganismsource.
 17. The method according to claim 1, wherein saidglycerophospholipid is de-oiled.
 18. The method according to claim 1,characterized in that said glycerophospholipid is effective at a lowerdosage compared to soybean-PS, while having similar and/or improvedbioactivity compared to soybean-PS.
 19. The method according to claim 1,wherein said omega-3 or omega-6 is more stable than an omega-3 oromega-6 in the free fatty acid form, bonded to a triglyceride or as anethyl ester.
 20. The method according to claim 1, wherein saidpharmaceutical or nutraceutical composition comprises said lipidpreparation at concentrations of 20% (w/w) PS and 30% (w/w) totalomega-3 LC-PUFA.
 21. The method according to claim 1, wherein said lipidis characterized by having a reduced or absent fish odor.
 22. The methodaccording to claim 1, wherein said nutraceutical composition is in theform of softgel capsules, tablets, syrups, or any other common dietarysupplement delivery system.
 23. The method according to claim 1, whereinsaid functional food is selected from dairy products, ice-creams,biscuits, soy products, bakery, pastry and bread, sauces, soups,prepared foods, frozen foods, condiments, confectionery, oils and fats,margarines, spreads, fillings, cereals, instant products, drinks andshakes, infant formulas, infant foods, bars, snacks, candies andchocolate products.
 24. The method according to claim 1, wherein saidpharmaceutical composition further comprises at least onepharmaceutically acceptable additive, diluent or excipient.
 25. Themethod according to claim 1, wherein said pharmaceutical compositionfurther comprises at least one pharmaceutically active agent.